Investigation of co-flow step emulsification (CFSE) microfluidic device and its applications in digital polymerase chain reaction (ddPCR).

HYPOTHESIS: The co-flow step emulsification (CFSE) is very sensitive to the two-phase fluid interfaces, we conjecture that the CFSE hydrodynamic model depends on several key factors and the droplet generation process can be precisely controlled, thus to obtain droplet emulsions with the "ultra-high volume fraction of inner-phase" and "flexible droplet size" characteristics. The resulting droplets are expected to be applied to droplet digital PCR (ddPCR) with "high information density" and "wide dynamic range" advances. EXPERIMENTS: By combining numerical simulation and fluid dynamics experiments, we have investigated the crucial parameters affecting the CFSE two-phase interface and finally achieved the prediction and guidance for CFSE droplet production. FINDINGS: With the help of the CFSE device, multivolume droplet populations were produced on demand. Then, ddPCR tests were performed with DNA concentrations from 10 copies/µL to 20,000 copies/µL. The CFSE device owns an ultra-wide dynamic range (up to 5 orders of magnitude), showing excellent quantification ability of nucleic acid targets.

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the DNA of the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of IGV gene subgroup-specific primers recognizing nearly all IGV genes together with primers for the J genes. By sequence analysis of IGV region genes from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D and J gene usage. Moreover, the presence and pattern of somatic IGV gene mutations give valuable insight into the differentiation stage of the B cells.

Expression Cloning of Antibodies from Single Human B Cells.

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in B-cell chronic lymphocytic leukemia (CLL) have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows for unbiased characterization of the human antibody repertoire on single-cell level through the generation of recombinant monoclonal antibodies from primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells to the RT-PCR-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information about the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g., by colony polymerase chain reaction (PCR) or restriction digest pattern analysis is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here, we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

Biofoundry-Assisted Golden Gate Cloning with AssemblyTron.

Golden Gate assembly is a requisite method in synthetic biology that facilitates critical conventions such as genetic part abstraction and rapid prototyping. However, compared to robotic implementation, manual Golden Gate implementation is cumbersome, error-prone, and inconsistent for complex assembly designs. AssemblyTron is an open-source python package that provides an affordable automation solution using open-source OpenTrons OT-2 lab robots. Automating Golden Gate assembly with AssemblyTron can reduce failure-rate, resource consumption, and training requirements for building complex DNA constructs, as well as indexed and combinatorial libraries. Here, we dissect a panel of upgrades to AssemblyTron's Golden Gate assembly capabilities, which include Golden Gate assembly into modular cloning part vectors, error-prone polymerase chain reaction (PCR) combinatorial mutant library assembly, and modular cloning indexed plasmid library assembly. These upgrades enable a broad pool of users with varying levels of experience to readily implement advanced Golden Gate applications using low-cost, open-source lab robotics.

The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Analysis of Cytokine-Inducible SH2-Containing Protein (CISH) in the Study of Immunosenescence.

The aging immune system undergoes significant changes, leading to a state known as immunosenescence. Understanding the molecular mechanisms underlying immunosenescence is crucial for developing targeted interventions to enhance immune functions in older individuals. This bio-protocol review focuses on the application of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the mRNA quantification of cytokine-inducible SH2-containing protein (CISH), an immune regulator overexpressed in T-cell responses from older adults. We outline a comprehensive protocol for the quantitative assessment of CISH mRNA expression, providing a valuable tool for researchers investigating immunosenescence.

An accurate haplotyping method using multiplex pyrosequencing with AS-PCR to detect ABCB1 haplotypes associated with rivaroxaban-derived hemorrhagic events.

In clinical practice, owing to the comprehensive genetic insights they offer, haplotypes have attracted greater attention than individual single nucleotide polymorphisms (SNPs). Due to the long distances across SNP locations, detecting the haplotype using genomic DNA is challenging. Current haplotyping methods are either expensive and labor-intensive (high-throughput DNA sequencing), or haplotyping a single clinical sample (computational approach) is impossible. Herein, we propose using mRNA as a haplotyping target to minimize the distance among SNPs and employing allele-specific PCR (AS-PCR) to pick up a desired haplotype, followed by multiplex pyrosequencing to type the alleles at the SNP location of interest. AS-PCR was improved by combining an additional 3'-phosphorylated modified probe to achieve the specific separation of two closely similar templates. Only the sample with more than two heterozygotes needs to be haplotyped; therefore, we propose a stratification strategy to screen the samples for further haplotyping. This method was evaluated by associating ABCB1 haplotypes with the rivaroxaban-derived side effect in a cohort of 505 patients with nephrotic syndrome, focusing on the SNPs of ABCB1: rs1236C > T, rs2677G > T/A, and rs3435C > T. We successfully identified five bleeding-related haplotypes: rs1236T-rs2677T-rs3435T, rs1236C-rs2677G-rs3435T, rs1236T-rs2677G-rs3435C, rs1236C-rs2677G-rs3435C, and rs1236T-rs2677T-rs3435C. We compared the results with those from the conventional computational algorithm PHASE and observed that PHASE results dismissed the impact of rs1236C-rs2677G-rs3435C and rs1236C-rs2677G-rs3435T on bleeding risk and erroneously suggested a false positive association of rs1236C-rs2677A-rs3435T with increased bleeding risk.

Prevalência de Influenza A, vírus sincicial respiratório eSARS-CoV-2 em pacientes com síndrome respiratóriaaguda grave em Passo Fundo, Rio Grande do Sul / Prevalence of Influenza A, respiratory syncytial virus andSARS-CoV-2 in patients with severe acute respiratorysyndrome in Passo Fundo, Rio Grande do Sul

A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.

Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.

Up-regulation of HOXA-AS2 and MEG3 long non-coding RNAs acts as a potential peripheral biomarker for bipolar disorder.

Bipolar disorder (BD) is a psychiatric condition that is frequently misdiagnosed and linked to inadequate treatment. Long non-coding RNAs (lncRNAs) have lately gained recognition as crucial genetic elements and are now regarded as regulatory mechanisms in the neurological system. Our objective was to measure the quantities of HOXA-AS2 and MEG3 ncRNA transcripts. HOXA-AS2 and MEG3 ncRNA levels were checked in the peripheral blood of 50 type I BD and 50 control samples by real-time PCR. Furthermore, we conducted ROC curve analysis and correlation analysis to examine the association between gene expression and specific clinical characteristics in instances with BD. Additionally, a computational study was performed to investigate the binding sites of miRNAs on the HOXA-AS2 and MEG3 lncRNAs. BD subjects showed a significant increase in the expression of HOXA-AS2 and MEG3 compared to controls. The lncRNAs HOXA-AS2 and MEG3 have an area under the ROC curve (AUC) values of 0.70 and 0.71, respectively. There was a significant correlation between the expression levels of ncRNAs HOXA-AS2 and MEG3 in the peripheral blood of patients with BD and occupation scores. The data presented indicate a potential correlation between the expression of HOXA-AS2 and MEG3 lncRNAs with an elevated risk of BD. Furthermore, these lncRNAs may be linked to several molecular pathways. Our findings indicate that the amounts of lncRNAs HOXA-AS2 and MEG3 in transcripts might be a promising potential biomarker for patients with BD.

Undiagnosed West Nile virus lineage 2d infection in a febrile patient from South-west Uganda, 2018.

We report the retrospective identification and subsequent recovery of a near-complete West Nile Virus lineage 2 genomes from a hospitalized patient with acute febrile illness in Uganda, using a combination of degenerate primer polymerase chain reaction screening and a novel 1200bp nanopore-based whole-genome amplicon sequencing scheme. This represents the first West Nile virus genome to be recovered from a human in Uganda since its discovery in 1937. Basic molecular rather than serological surveillance methods could be more widely deployed in the region to better diagnose febrile infections.

Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolated From Diabetic Foot Ulcers in a Tertiary Care Hospital in Mysuru, South India.

Background Diabetic foot ulcers (DFUs) are common complications in diabetes patients, often leading to sepsis and leg amputation. Methicillin-resistant Staphylococcus aureus (MRSA) infections in DFUs pose challenges due to methicillin resistance with mecA and mecC genes. This study aims to assess the prevalence of MRSA in clinical isolates from DFUs, analyze the antibiogram of MRSA isolates, and detect the presence of the mecA and mecC genes among MRSA isolates. Methodology The isolated S. aureus colonies were identified and antimicrobial susceptibility was performed using the Vitek-2 Compact system. Methicillin resistance was also confirmed through the disc diffusion method. Confirmed methicillin-resistant isolates were subjected to real-time polymerase chain reaction (RT-PCR) to detect mecA and mecC genes. Results A total of 474 purulent samples from DFUs yielded 541 distinct isolates, comprising 201 gram-positive and 340 gram-negative organisms. Among the gram-positive organisms, Staphylococcus species predominated, with 79 S. aureus isolates, 34 of which were methicillin-resistant. All MRSA isolates (100%) were sensitive to tetracycline, linezolid, teicoplanin, and vancomycin, and 94% were sensitive to cotrimoxazole but least susceptible to ciprofloxacin and levofloxacin. RT-PCR confirmed the presence of mecA genes in all 34 isolates and mecC genes in three isolates. Conclusions The presence of mecA in all 34 MRSA isolates underscores consistent methicillin resistance. The co-occurrence of mecA and mecC in three isolates hints at genetic diversity. Two MRSA isolates positive for mecC were isolated from rural patients involved in farming and animal husbandry, suggesting an occupational risk. The third patient was from a non-rural area, indicating potential alternative transmission pathways warranting further investigation.

Corrigendum: Study the effect of Enterobacter cloacae on the gene expression, productivity, and quality traits of Curcuma longa L. Plant.

[This corrects the article DOI: 10.3389/fpls.2024.1393198.].

Upper respiratory tract detection of Mycoplasma ovipneumoniae employing nasopharyngeal swabs.

BACKGROUND: Flock-level prevalence and characterization of Mycoplasma ovipneumoniae is determined almost exclusively using nasal swabbing followed by molecular detection with either quantitative PCR or multi-locus sequence typing. However, the diagnostic performance and efficiency of swabbing the nasal passage compared to other anatomical locations has not been determined within sheep populations. The goal of this research was to assess the diagnostic capability of nasopharyngeal swabs in comparison to nasal swabs for the detection of Mycoplasma ovipneumoniae. RESULTS: Nasal and nasopharyngeal swabs were collected during a controlled exposure study of domestic sheep with Mycoplasma ovipneumoniae. Both swab types were then analyzed via conventional and quantitative PCR. This dataset showed that the use of nasopharyngeal swabs in lieu of nasal swabs resulted in higher sensitivity, reduced inhibition during quantitative PCR, and higher bacterial copy numbers per swab. Moreover, it was demonstrated that diagnostic sensitivity could be further increased during quantitative PCR via ten-fold dilution of the extracted DNA. To confirm these observations in naturally infected animals, we conducted a field study employing a production flock of domestic sheep using both nasal and nasopharyngeal swabbing techniques. Extracted DNA was assessed using the same molecular techniques, where detection of Mycoplasma ovipneumoniae was confirmed by sequencing of either the rpoB or 16S rRNA gene. Similar improvements were observed for nasopharyngeal swabs and template treatment methods within the naturally infected flock. CONCLUSIONS: Results demonstrate increased diagnostic sensitivity and specificity when sampling with nasopharyngeal swabs as compared to nasal swabs. Therefore, alternate field-testing strategies employing nasopharyngeal swabs should be considered for diagnosis of the presence of M. ovipneumoniae. Importantly, sample treatment following acquisition was found to affect the sensitivity of quantitative PCR, where dilution of eluted DNA template doubled the calculated sensitivity. This demonstrates that, in addition to anatomical location, the presence of inhibitory components in swab extracts also strongly influences diagnostic performance.

International external quality assessment study for detection of monkeypox virus by PCR supporting laboratory preparedness during the 2022-2023 mpox outbreak and beyond.

BACKGROUND: Diagnostic capabilities and correspondent External Quality Assessments (EQA) are key for outbreak preparedness. To support diagnostic facilities with a quality assessment of newly established monkeypox virus (MPXV) molecular diagnostic workflows, Quality Control for Molecular Diagnostics (QCMD) and the Bundeswehr Institute of Microbiology (IMB) piloted an international EQA study conducting four challenges from autumn 2022 to summer 2023 during the global mpox outbreak. OBJECTIVES: To assess the performance (sensitivity/specificity) of molecular assays used by diagnostic laboratories. STUDY DESIGN: Inactivated EQA panels were prepared and distributed containing seven samples of clade Ia and clade IIb MPXV strains at different viral concentrations, two specificity controls with other zoonotic orthopoxviruses (vaccinia and cowpox virus) and a negative control. Assessment was based on reported qualitative testing results. RESULTS: In this outbreak-related EQA study, a total of 192 laboratories from 37 countries reported 346 qualitative datasets. Overall, core samples were correctly detected by approximately 92 % of participants in all four challenges. While sensitivity performance was acceptable in at least 90 % of datasets correctly reported even for educational MPXV-positive samples with low viral concentration [102 genome equivalents (GE)/mL], several laboratories reported the educational specificity controls as false positives or were unable to differentiate MPXV from related orthopoxviruses. CONCLUSIONS: Mpox is now a globally occurring infection with a demand for quality-assured diagnostic capabilities. The newly established EQA scheme presented here, offers a multi-purpose panel for orthopoxviruses with a focus on MPXV which will continue to ensure diagnostic quality in clinical settings with up-to-date sample panels.

The Distribution of Respiratory Viral Pathogens Among the Symptomatic Respiratory Tract Infection Patients From Dhaka City in the Pre-COVID-19 Pandemic Era.

Rapid and accurate diagnosis is crucial for determining the etiology and, perhaps, effectively treating and preventing viral respiratory infections. A multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay⁠⁠⁠⁠⁠⁠⁠ was utilized to determine the prevalence of viral etiology in cases of acute respiratory tract infections (ARTIs). Outpatient department (OPD) and intensive care unit (ICU) patients with fever and respiratory symptoms were enrolled from December 2018 to April 2020. Nucleic acids were extracted from the respiratory tract samples using the SV Total RNA Isolation System (Promega Corporation, Madison, WI), and virus identification was performed using qRT-PCR assay (Fast Track Diagnostics {FTD} Respiratory Pathogens, Esch-sur-Alzette, Luxembourg). A total of 152 samples were collected from OPD and ICU. Among them, 32.23% (n = 49) of the patients were positive for at least one respiratory virus. From 49 infected cases, 42 had only a single viral pathogen, whereas seven had co-infections. Of the patients, 32.25% (30) in the OPD and 32.20% (19) in the ICU tested positive for the respiratory viral pathogen. Among the OPD patients, human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1 were detected as predominant viruses (10.75%), followed by influenza virus (IFV) (8.6%), human rhinoviruses (HRVs) (6.45%), human parainfluenza viruses (HPIVs) (6.45%), respiratory syncytial virus (RSV) (3.22%), and adenovirus (2.15%). In ICU cases, HPIV and HRV were detected as predominant viruses (8.47% each), followed by HCoV (5.08%), human metapneumovirus (HMPV) (5.08%), influenza A virus (IAV) (3.38%), adenovirus (3.38%), and RSV (1.69%). This study highlighted the prevalence of respiratory viruses in both the community and hospital settings during pre-COVID-19, indicating a significant presence among patients in these environments.

Comparative evaluation of real-time polymerase chain reaction, conventional PCR and microscopy for detection of Babesia bigemina in bovines of Punjab (India).

Babesia bigemina is a major cause of bovine babesiosis, an economically devastating tick-borne disease that needs timely diagnosis for precise treatment. In present investigation, the detection efficacy of real-time PCR (qPCR) in comparison to conventional PCR and microscopy targeting 18 S ribosomal gene was evaluated on 95 bovines (70 cattle and 25 buffaloes) suspected for babesiosis. Real-time PCR was standardized with the 10-fold serial dilutions in duplication of the given positive control (2 × 106 copy number) ranging from 106 to 100 copy number/µL and mean Ct value of each dilution was taken to extrapolate the curve. The samples with Ct value 36.92 of 10 copy number/µL were considered as positive. Out of 95 samples, 5 (5.26%), 21 (22.10%) and 49 (51.58%) positive by microscopy, conventional PCR and real-time PCR were in corresponding range of > 106-104, 104-103, and 103-<10 copy number/µL, respectively. The concordance of real-time PCR with conventional PCR and microscopy was moderate (Kappa = 0.523) and slight (Kappa = 0.09), respectively. The cows were at four times risk than the buffaloes for B. bigemina infection (Odds ratio:3.85, CI:1.4255-10.4370). This pioneer report from Punjab state (India) of application of real-time PCR to detect B. bigemina in bovines was found to be more sensitive than conventional PCR and microscopy that needs further investigations on a greater number of random samples.

Circulating miRNA-106b-5p As a Potential Biomarker for Coronary Artery Disease.

Coronary artery diseases (CAD) represent a significant global health concern and are recognized as a primary contributor to mortality on a worldwide scale. Early diagnosis of CAD is one of promising goal to manage this disorder. Recent investigations have highlighted the pivotal involvement of microRNAs (miRNAs) in diverse health conditions, notably CAD. The principal objective of this investigation was to identify appropriate miRNAs that could be employed for the early detection of CAD. In the present study, we analyzed dataset of CAD (GSE113079) and 100 differentially expressed mRNAs (DEmRNAs) were detected. The miRNAs that have a significant interaction with DEmRNAs were chosen. By computational prediction method, 5 miRNAs (miR-106b-5p, miR-20a-3p, miR-17-3p, miR-146a-5p, and miR-155-3p) were selected. Finally, we assessed the anticipated expression levels of microRNAs in CAD patients and healthy control groups. Our findings revealed a statistically significant elevation solely in the expression level of miR-106b-5p within the CAD group when compared to the control group (p>0.001). Our study demonstrated an elevation in the expression of miR-106b-5p in individuals diagnosed with CAD. This microRNA may be used as a diagnostic biomarker in patients with CAD. However, further investigations are needed to confirm these results.

Unlocking Therapeutic Potential: Enhanced shRNA Delivery with Tat Peptide in the Human Respiratory Syncytial Virus Treatment.

Purpose: This research investigated the development of short hairpin RNA (shRNA) molecules designed to target specific regions of the human respiratory syncytial virus (HRSV) M and F genes. The study aimed to assess the therapeutic potential of these shRNAs and evaluate the effectiveness of Tat peptide-mediated delivery in enhancing their functionality. Methods: We acquired isolates from pediatric patients experiencing respiratory illness then cultured in HEp-2 cells. We constructed plasmids expressing shRNAs. Tat peptide as a facilitator for shRNA plasmid delivery was used. The cytotoxicity of ribavirin, shRNA constructs, and control agents was assessed using the MTT assay. The transfection efficiency of Tat peptide-mediated shRNA delivery with that of lipofectamine 3000TM were compared. Finally, real-time PCR was employed to quantify HRSV replication in the treated cells. Results: Tat peptide-mediated delivery of shRNA plasmids significantly suppressed the expression of the M and F genes of HRSV compared to lipofectamine 3000TM. This suppression was evident in both short-term experiments and scenarios involving stable shRNA expression. Furthermore, the combination of ribavirin with shRNA treatment resulted in a substantial reduction in viral load. Notably, the most pronounced antiviral effect was observed when both shRNAs were employed simultaneously. Conclusion: Our findings suggest that Tat peptide-mediated delivery of shRNA plasmids holds significant potential for achieving stable suppression of HRSV genes. This approach warrants further investigation as a potential gene therapy strategy for HRSV. By demonstrating promising results in vitro, this study highlights the need for future in vivo studies to comprehensively evaluate the therapeutic potential of this approach in a clinical setting.